Despite considerable efforts to understand the causes underlying the severe neuro-toxicity of methyl mercury (MeHg), the specific biochemical mechanisms by which methyl mercury (MeHg) impairs neurological function have not been revealed. In an attempt to identify biochemical loci targeted by MeHg we have utilized cerebellar granule neurons, a cell type which is exceptionally sensitive to MeHg in vivo. In cell culture, the system allows prolonged exposure to low concentrations of MeHg while manifesting several neurodevelopmental characteristics such as neurite elongation and synaptogenesis. We have found that cultured granule neurons display extremely high sensitivity to MeHg. One of the most important biochemical regulatory pathways considered to be crucial to nervous system function is that of protein phosphorylation. We have observed a stimulation of up to 1.7-fold in phosphorylation of total protein following 24 h exposure to 3 uM MeHg. At this dose glial cultures manifested significant inhibition of phosphorylation, suggesting that MeHg- induced stimulation of phosphorylation is a neuron-specific event. The goal of our proposed research is to characterize the mechanism of this profound stimulation by analyzing the effects of MeHg on 1) protein kinase enzymes, 2) second messengers and 3) phosphoprotein phosphatase. In addition, we plan to evaluate the specific contribution of aberrant protein phosphorylation to neuronal cytotoxicity and compare these toxic effects with those produced by MeHg. Two specific phosphorylation events will be explored in depth with respect to the impact of MeHg. 1) Phosphorylation of the cytoskeletal proteins, tubulin and tau factor, undoubtedly plays a major role in neurite outgrowth and function and MeHg-induced impairment of these reactions may underlie neuritic malformations observed as an early pre-toxic response to MeHg. 2) Expression of a group of phosphoproteins, pp32/pI 5.7-6.1, is greatly enhanced in MeHg-treated cultures and may reflect a specific stress response of granule neurons to toxic heavy metals. The nature of these proteins as well as the kinases responsible for their phosphorylation will be investigated. The end result of these studies will be a significantly deeper understanding of protein phosphorylation in the cerebellar granule cell and the aberrations induced by prolonged exposure to low levels of MeHg.